Synthetic peptides reducing or removing bags formed under the lower eye contour and their use in cosmetic or dermopharmaceutical compositions

ABSTRACT

The invention relates to peptides of general formula (I): 
                         
that can reduce or remove bags formed under the eyes, their stereoisomers and racemic or non-racemic mixtures thereof, and the cosmetically or dermopharmaceutically acceptable salts thereof, wherein X is cystenyl, seryl, threonyl or aminobutyryl; R 1  is H or alkyl, aryl, aralkyl or acyl group; and R 2  is amino, hydroxy or thiol, all of them substituted or non-substituted with aliphatic or cyclic groups. The invention also relates to a method of obtaining, cosmetic or dermopharmaceutical compositions containing them and their use for treating skin, preferably for reducing or removing bags formed under the eyes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of PCT PatentApplication No. PCT/ES2005/00514, Filed on Sep. 22, 2005, which claimsthe benefit of Spain Patent Application P200402364, filed on Oct. 5,2004.

FIELD OF THE INVENTION

The present invention relates to synthetic peptides reducing or removingbags formed under the eyes and to cosmetic or dermopharmaceuticalcompositions containing said peptides useful in the treatment of skin,preferably facial skin, and especially the skin located under the eyes,with the aim of reducing or removing the swelling as well as improvingits firmness, hydration and elasticity.

BACKGROUND OF THE INVENTION

The appearance of bags in the lower eye contour is a common cosmeticproblem occurring when the skin of the lower eyelid is slightly swollenand hangs. The skin surrounding the eyes is relatively thin and has lessoily composition than many other areas of the skin. For this reason,aging, stress, different illnesses and environmental pollution can showtheir first symptoms with a swelling of the lower eyelids and theappearance of bags due to the loss of firmness and elasticity of theskin which is located under the eyes. Fluid accumulation under the skinin the area located under the eyes gives rise to an edema which is shownas swollen eyes, frequently darker in comparison to surrounding facialareas (“dark circles”), which the consumer perceives as unacceptable oranti-aesthetic.

Up until now, the exact reasons for which anti-aesthetic under-eye bagsare formed are not known, but different external factors such as stress,excessive caffeine or alcohol consumption and lack of sleep have beenidentified as associated factors or inducers of the problem. Likewise,the literature has described internal factors contributing to theformation of under-eye bags such as kidney malfunction and fluidretention, high blood pressure, inflammation, allergic components(allergic rhinitis) or lymphatic drainage alteration. Intrinsic skinaging, orbicular muscle relaxation as well as damage caused byultraviolet radiation must also be considered together with thesefactors.

As the skin loses its elasticity and the muscles weaken with age,flaccid skin can accumulate around the eyes, forming folds in theeyelids. Furthermore, the fat accommodating and supporting the eyes intheir sockets tends to move towards the outside of the eye cavities andto accumulate around the eyes in the form of bulging eyes. Swollen orbulging eyelids can be due to an accumulation of fat in the orbiculararea as well as to an accumulation of flaccid skin of said area.

With the aim of recovering a young and a non-fatigued appearance of thefacial expression, cosmetic surgery is frequently used to removeunder-eye bags (blepharoplasty), in a process consisting of makinginternal and external incisions in the eyelids with the aim of removingthe excess accumulated fat and/or skin. Blepharoplasty is currently theprocess that is most frequently carried out by plastic surgeons in theUnited States [Castro, E. and Foster, J. A. (1999) Upper lidblepharoplasty Facial Plast. Surg. 15 (3), 173-181]. However, despitethe fact that said surgery is considered to be a minor surgical process,it is a technique that is not risk-free, due to the associated risks ofthe anesthesia used in the intervention as well as the risks ofpotential post-operative infections. Therefore, there is still a need tofind a simple, effective and risk-free solution for reducing andremoving under-eye bags.

DESCRIPTION OF THE INVENTION

The present invention provides a simple, effective and risk-freesolution for reducing or removing the under-eye bags, comprising thedevelopment of synthetic peptides that can reduce or remove under-eyebags, as well as improve the firmness and elasticity of the skin of thelower eyelid area.

Therefore, a first aspect of this invention relates to a peptide thatcan reduce or remove bags formed under the eyes, according the generalformula (I):

its stereoisomers and racemic or non-racemic mixtures thereof, and thecosmetically or dermopharmaceutically acceptable salts thereof, wherein:X can be: cystenyl cysteinyl, seryl, threonyl or aminobutyryl;R₁ can be: H or alkyl, aryl, aralkyl or acyl group; andR₂ can be: amino, hydroxy or thiol, all of them substituted ornon-substituted with aliphatic or cyclic groups.

The preferred structures of the peptides represented in the generalformula (I) are those wherein:

X can be: seryl or aminobutyryl;

R₁ can be: H or saturated or unsaturated, branched or cyclic, linear C₂to C₂₄ acyl; and

R₂ can be: amino or hydroxy, substituted or non-substituted withsaturated or unsaturated, branched or cyclic, linear, aliphatic C₁ toC₂₄ groups.

The peptides of the present invention can exist as stereoisomers ormixtures of stereoisomers; for example, the amino acids forming them canhave the L-, D-configuration or can be racemic independently of oneanother. It is therefore possible to obtain isomeric mixtures as well asracemates or diastereomeric mixtures, or pure diastereomers orenantiomers, depending on the number of asymmetric carbons and of whichisomers or isomeric mixtures are present.

The preferred structures of the peptides of general formula (I) are pureisomers, i.e., enantiomers or diastereomers.

In the context of the present invention, the term “aliphatic group”relates to a saturated or unsaturated, linear or cyclic hydrocarbongroup.

The term “hydrocarbon group” is used in the present invention to includealkyl, alkenyl and alkinyl alkynyl groups for example.

The term “alkyl group” relates to a linear or branched, saturatedhydrocarbon group, including for example, methyl, ethyl, isopropyl,isobutyl, t-butyl, heptyl, dodecyl, hexadecyl, octadecyl, amyl,2-ethylhexyl, 2-methylbutyl, 5-methylhexyl and the like.

The term “alkenyl group” relates to a linear or branched, unsaturatedhydrocarbon group, with one or more carbon-carbon double bonds, such asthe vinyl group.

The term “group alkinylalkynyl group” relates to a linear or branched,unsaturated hydrocarbon group, with one or more carbon-carbon triplebonds.

The term “cyclic group” relates to a closed hydrocarbon ring, which canbe classified as alicyclic, aromatic or heterocyclic group.

The term “alicyclic group” relates to a cyclic hydrocarbon group withproperties similar to aliphatic groups.

The term “aromatic group” or “aryl group” relates to a mono orpolycyclic aromatic hydrocarbon group.

The term “heterocyclic group” relates to a closed hydrocarbon ring, inwhich one or more of the ring atoms is an element other than carbon (forexample, nitrogen, oxygen, sulfur, etc.).

As understood in this technical area, the existence of high degree ofsubstitution is not only tolerated but recommended. Therefore, there maybe substitution in the peptides of the present invention. For thepurpose of simplifying the present description of the invention, theterms “group” and “block” will be used to distinguish between chemicalspecies allowing substitution or which can be substituted (“group”), andthose not allowing substitution or which cannot be substituted(“block”). In this way, when the term “group” is used to describe achemical substituent, the described chemical material includes both thenon-substituted group and that containing O, N or S atoms.

On the other hand, when the term “block” is used to describe a chemicalcompound or substituent, only non-substituted chemical material can beincluded. For example, the expression “alkyl group” will not onlyinclude open-chain saturated alkyl substituents such as methyl, ethyl,propyl, isobutyl and the like, but also alkyl substituents containingother substituents known in the state of the art, such as hydroxy,alcoxy, amino, carboxyl, carboxamido, halogen atoms, cyano, nitro,alkylsulfonyl, and others. In this way, “alkyl group” includes ether,haloalkyl, alcohol, thiol, carboxyl, amine, hydroxyalkyl, sulfoalkyl,guanidine groups and others. On the other hand, the expression “alkylblock” is only limited to the inclusion of open-chain saturated alkylsubstituents such as methyl, ethyl, propyl, isobutyl and the like.

The cosmetically or dermopharmaceutically acceptable salts of thepeptides of formula (I) provided by this invention are included withinthe scope of the present invention. The term “cosmetically ordermopharmaceutically acceptable salts” includes the salts usually usedto form metal salts or acid addition salts, either organic (such asacetate, citrate, oleate, oxalate or gluconate among others) orinorganic (such as for example chloride, sulfate, borate or carbonateamong others). The nature of the salt is not critical, provided that itis cosmetically or dermopharmaceutically acceptable. The cosmetically ordermopharmaceutically acceptable salts of the peptides of formula (I)can be obtained by conventional methods, well known in the state of theart.

The synthesis of the peptides of general formula (I) can be carried outaccording to conventional methods known in the state of the art, such asfor example the adaptation of solid-phase peptide synthesis methods[Stewart J. M. and Young J. D. (1984) Solid Phase Peptide Synthesis, 2ndedition, Pierce Chemical Company, Rockford, Ill. Bodanzsky M. andBodanzsky A. (1984) The practice of Peptide Synthesis, Springer Verlag,New York. Lloyd-Williams, P., Albericio, F. and Giralt, E. (1997)Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, BocaRaton (Fla., USA)], solution synthesis, a combination of solid-phasesynthesis and solution synthesis methods or enzymic methods [Kullmann W(1980) Proteases as catalysts for enzymic syntheses of opioid peptidesJ. Biol. Chem. 255, 8234-8238]. The peptides can also be obtained by thefermentation of a bacterial strain that is modified or unmodified bygenetic engineering with the aim of producing the desired sequences.

For example, a method for obtaining peptides of general formula (I) isthat in which a fragment of the peptide of general formula (I), having afree carboxyl group or a reactive derivative thereof, is reacted with acomplementary fragment, having an amino group, with at least one freehydrogen atom, with the subsequent formation of an amide type bond, andwherein the functional groups of said fragments that do not participatein the formation of the amide-type bond, if they exist, are convenientlyprotected with temporary or permanent protective groups.

Another example of a method for obtaining of peptides of general formula(I) is that in which a fragment of the peptide of general formula (I)having a leaving group, such as for example the tosyl group, the mesylgroup and halogen groups among others, is reacted with a complementaryfragment having an amino group with at least one free hydrogen atom bymeans of a nucleophilic substitution reaction, and wherein saidfunctional groups of the fragments that do not participate in theformation of the N—C bond, if they exist, are conveniently protectedwith temporary or permanent protective groups. Examples of protectivegroups, their introduction and elimination are described in theliterature [Greene T. W. (1981) Protective groups in organic synthesis,John Wiley & Sons, New York. Atherton B. and Sheppard R. C. (1989) SolidPhase Peptide Synthesis: A practical approach, IRL Oxford UniversityPres]. The term “protective groups” also includes polymeric supportsused in solid-phase synthesis.

When the synthesis is carried out completely or partially in solidphase, the following can be mentioned as solid supports to be used inthe method of the invention: supports made of polystyrene, polyethyleneglycol-grafted polystyrene and the like, such as for examplep-methylbenzhydrylamine resins (MBNA) [Matsueda G. R. and Stewart J. M.(1981) A p-methylbenzhydrylamine resin for improved solid-phasesynthesis of peptide amides Peptides 2, 45-50.], 2-chlorotrityl resins[(a) Barlos K., Gatos D., Kallitsis J., Papaphotiu G., Sotiriu P.,Wenqing Y. and Schäfer W (1989) Darstellung geschützter peptid-fragmenteunter einsatz substituierter triphenylmethylharze Tetrahedron Lett. 30,3943-3946. (b) Barlos K., Gatos D., Kapolos S., Papaphotiu G., SchäferW. and Wenqing Y. (1989) Veresterung von partiell geschütztenpeptide-fragmenten mit harzen. Einsatz von 2-chlortritylchlorid zursynthese von Leu 15-gastrin I Tetrahedron Lett. 30, 3947-3951],TentaGel® resins and the like, which may or may not include a labilespacer such as 5-(4-aminomethyl-3,5-dimethoxyphenoxy) valeric acid (PAL)[Albericio F., Kneib-Cordonier N., Biancalana S., Gera L., Masada R. I.,Hudson D. and Barany G. (1990) Preparation and application of the5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)-valericacid (PAL) handle for the solid-phase synthesis of C-terminal peptideamides under mild conditions J. Org. Chem. 55, 3730-3743],2-[4-aminomethyl-(2,4-dimethoxyphenyl)phenoxyacetic acid (AM) [Rink H.(1987) Solid-phase synthesis of protected peptide fragments using atrialkoxy-diphenyl-methylester resin Tetrahedron Lett. 28, 3787-3790],Wang [Wang, S. S. (1973) p-Alkoxybenzyl Alcohol Resin andp-Alkoxybenzyloxycarbonylhydrazide Resin for Solid Phase Synthesis ofProtected Peptide Fragments J. Am. Chem. Soc. 95, 1328-1333] and thelike, allowing the deprotection and simultaneous cleavage of thecompound from the polymeric support.

The peptides according to the invention can form part of several typesof compositions for their external application in a body of a mammal,preferably a human being. In this sense, the invention provides acosmetic or dermopharmaceutical composition comprising peptides ofgeneral formula (I). Said compositions can be prepared by conventionalmethods known by persons skilled in the art.

The peptides object of the invention have a variable water-solubility,according to the nature of the R₁, R₂, and X groups. Those which are notwater-soluble can be solubilized in conventional cosmetically ordermopharmaceutically acceptable solvents such as for example ethanol,propanol or isopropanol, propylene glycol, glycerin, butylenes glycol orpolyethylene glycol. The peptides can also be previously incorporated incosmetic carriers such as liposomes, milliparticles, microparticles andnanoparticles as well as in sponges, millispheres, microspheres andnanospheres, millicapsules, microcapsules and nanocapsules.

These preparations can be used in different types of formulations suchas for example, creams, lotions, gels, oils, liniments, serums, mousses,ointments, bars, pencils or sprays, including “leave on” and “rinse-off”formulations, and can also be incorporated by means of techniques knownby persons skilled in the art to different types of solid accessoriessuch as towelettes, hydrogels, adhesive (or non-adhesive) patches orface masks, or can be incorporated to different make-up line productssuch as concealers, make-up foundations, lotions, make-up removallotions among others.

The compositions mentioned in the present invention can containadditional ingredients commonly used in compositions for the care andtreatment of skin, such as for example and in a non-limiting sense,emulsion agents, emollients, organic solvents, skin conditioners such asfor example, humectants, alpha hydroxy acids, moisturizers, vitamins,pigments or dyes, gelling polymers, thickeners, softeners, anti-wrinkleagents, whitening agents, compounds capturing free radicals,anti-oxidizing compounds, compounds stimulating the synthesis of dermalor epidermal macromolecules and/or able to prevent their degradation,such as for example compounds stimulating collagen synthesis, compoundsstimulating elastin synthesis, compounds inhibiting collagendegradation, compounds stimulating fibroblast proliferation, compoundsstimulating keratinocyte proliferation, compounds stimulatingkeratinocyte differentiation, skin relaxing compounds, compoundsstimulating glycosaminoglycan synthesis, firming compounds, compoundsacting on capillary circulation, compounds acting on cell metabolism,compounds stimulating melanin synthesis, preservatives, perfumes,chelating agents, plant extracts, essential oils, marine extracts, cellextracts and sunscreens (organic or mineral photoprotection agents thatare active against ultraviolet A and B rays), among others, providedthat they are physically and chemically compatible with the rest of thecomponents of the composition and especially with the peptides of thepresent invention.

The compositions of the present invention can contain or becoadministered with analgesic compounds and/or anti-inflammatorycompounds for the purpose of reducing the swelling and irritationassociated to under-eye bags. Steroid type compounds such ashydrocortisone or natural extracts or essential oils with intrinsicanti-inflammatory and analgesic activity can be emphasized among thesecompounds.

The peptides of general formula (I) are used in the cosmetic ordermopharmaceutical compositions of the present invention atcosmetically or dermopharmaceutically effective concentrations toachieve the desired effect; preferably between 0.00001% (by weight) and10% (by weight); preferably between 0.0001% (by weight) and 5% (byweight) and more specifically between 0.001% (by weight) and 1% (byweight).

Therefore, an additional aspect of this invention relates to the use ofpeptides of general formula (I) in the manufacture of a cosmetic ordermopharmaceutical composition for the treatment of skin, preferablyfacial skin and more specifically for reducing or removing under-eyebags.

The present invention further provides a cosmetic or dermopharmaceuticalmethod for reducing or removing bags formed under the eyes in humans,comprising the administration of an effective amount of peptides ofgeneral formula (I), preferably in the form of a cosmetic ordermopharmaceutical composition containing it.

EXAMPLES

The following specific examples provided herein are useful forillustrating the nature of the present invention. These examples areincluded solely for illustrative purposes and must not be interpreted aslimitations to the invention claimed herein.

General Methodology

Chemical Synthesis

All the synthetic processes are carries out in polypropylene syringesequipped with porous polyethylene disks. All the reagents and solventsare of a quality for synthesis and are used without any additionaltreatment. The elimination of the Fmoc group is carried out withpiperidine-DMF (2:8, v/v) (1×1 minutes, 1×5 minutes; 5 mL/g resin)[Lloyd-Williams, P., Albericio, F. and Giralt, E. (1997) ChemicalApproaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton(Fla., USA)]. The washings between the steps of deprotection, couplingand, once again, deprotection have been carried out with DMF (3×1minutes) using 10 mL of solvent/g of resin each time. The couplingreactions have been carried out with 3 mL of solvent/g of resin. Thecontrol of the couplings is carried out by means of the ninhydrin test[Kaiser, E., Colescott R. L., Bossinger C. D. and Cook P. I. (1970)Color test for detection of free terminal amino groups in thesolid-phase synthesis of peptides Anal. Biochem. 34, 595-598]. All thesynthetic transformations and washings have been carried out at 25° C.

The chromatographic analysis by HPLC was carried out in Shimadzuequipment (Kyoto, Japan) using a reversed-phase column thermostatted at30° C. (250×4.0 mm, Kromasil C₈, 5 μm, Akzo Nobel, Sweden). The elutionwas carried by means of a gradient of acetonitrile (+0.07% TFA) in water(+0.1% TFA) at a flow of 1 mL/minutes and the detection is carried outat 220 nm.

Abbreviations

The abbreviations used for the amino acids follow the rules of theIUPAC-IUB Commission on Biochemical Nomenclature specified in Eur. J.Biochem. (1984) 138, 9-37 and in J. Biol. Chem. (1989) 264, 633-673.

Abu, 2-aminobutyric acid; βAla, beta-alanine, 3-aminopropionic acid; AM,2-[4-aminomethyl-(2,4-dimethoxyphenyl)-phenoxyacetic acid; Boc,tert-butyloxycarbonyl; DCM, dichloromethane; DIEA,N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMF,N,N-dimethylformamide; ES-MS, electrospray mass spectrometry; Fmoc,fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HPLC, highperformance liquid chromatography; MBHA, resin p-methylbenzhydrylamine;MeCN, acetonitrile; MeOH, methanol; PAL,5-(4-aminomethyl-3,5-dimethoxyphenoxy) valeric acid; Palm, palmitoyl;tBu, tert-butyl; THF, tetrahydrofuran; TFA, trifluoroacetic acid; Trt,trityl.

Example 1 Obtaining Ac-βAla-His-Cys-His-OH

5.5 g of Fmoc-L-His(Trt)-OH (8.9 mmol, 1 equiv) dissolved in 55 mL ofDCM to which 1.3 mL of DIEA (2.9 mmol, 0.33 equiv) have been added wereincorporated to dry 2-chlorotrityl resin (5.5 g, 8.8 mmol). It was leftstirring for 5 minutes, after which 2.5 mL of DIEA (5.9 mmol, 0.67equiv) were added. It was allowed to react for 40 minutes. The remainingchloride groups were blocked by treatment with 4.4 mL of MeOH.

The amino terminal Fmoc group was deprotected as described in generalmethods and 12.89 g of Fmoc-L-Cys (Trt)-OH (22 mmol, 2.5 equiv) wereincorporated to the peptidyl-resin in the presence of DIPCDI (3.39 mL,22 mmol, 2.5 equiv) and HOBt (3.37 g, 22 mmol, 2.5 equiv) using DMF as asolvent for 1 hour. The resin was subsequently washed as described ingeneral methods and the treatment for deprotecting the Fmoc group wasrepeated to incorporate the next amino acid. By following the describedprotocols, 13.63 g of Fmoc-L-His(Trt)-OH (22 mmol, 2.5 equiv) and 6.85 gof Fmoc-βAla-OH (22 mmol, 2.5 equiv) were coupled sequentially with thepresence in each coupling of 3.37 g of HOBt (22 mmol, 2.5 equiv) and3.39 mL of DIPCDI (22 mmol, 2.5 equiv).

The N-terminal Fmoc group was deprotected as described in generalmethods, the peptidyl-resin was treated for 30 minutes with aceticanhydride (2.1 mL, 22 mmol) in the presence of DIEA (7.53 mL, 22 mmol)using DMF as a solvent, it was washed with DMF (5×1 minutes), DCM (4×1minutes), diethyl ether (4×1 minutes) and dried under vacuum.

12.36 g of the dry peptidyl-resin was treated with 87 mL ofTFA-^(i)Pr₃Si—H₂O (90:5:5) for 2 hours at room temperature. Thefiltrates were collected on cold diethyl ether (700 mL), it was filteredthrough porous plate and the precipitate was washed 5 times with ether(500 mL). The final precipitate was dried under vacuum.

The analysis by HPLC in a gradient from 2 to 32% of MeCN (+0.07% TFA) inH₂O (+0.1% TFA) indicated a retention time of 12.63 minutes and a puritygreater than 85%. Its molecular weight was determined by ES-MS [(M+H)⁺_(theoretical) 509.19, (M+H)⁺ _(exp) 509.2].

Example 2: (Prophetic) Synthesis of Palm-βAla-His-Ser-His-NH₂

0.685 mg of the Fmoc-AM-MBHA resin with a functionalization of 0.73mmol/g (0.5 mmol) was treated with piperidine-DMF according to thedescribed general protocol for the purpose of eliminating the Fmocgroup. 1.58 g of Fmoc-L-His(Trt)-OH (2.5 mmol, 5 equiv) wereincorporated to the resin in the presence of DIPCDI (385 μL, 2.5 mmol, 5equiv) and HOBt (385 mg, 2.5 mmol, 5 equiv) using DMF as a solvent for 1hour.

The resin was subsequently washed as described in the general methodsand the treatment for deprotecting the Fmoc group was repeated toincorporate the next amino acid. By following the described protocols,0.95 g of Fmoc-L-Ser (tBu)-OH (2.5 mmol, 5 equiv), 1.59 g ofFmoc-L-His(Trt)-OH (2.5 mmol, 5 equiv) and 0.77 g of Fmoc-βAla-OH (2.5mmol, 5 equiv) were sequentially coupled with the presence in eachcoupling of 385 mg of HOBt (2.5 mmol, 5 equiv) and 385 μL of DIPCDI (2.5mmol, 5 equiv).

The N-terminal Fmoc group was deprotected as described in generalmethods, and 1.28 g of palmitic acid (5 mmol, 10 equiv) pre-dissolved inDMF (10 mL) were incorporated in the presence of 770 mg of HOBt (5 mmol,10 equiv) and 770 μL of DIPCDI (5 mmol, 10 equiv). It was left reactingfor 15 hours, after which the resin was washed with THF (5×1 min), DCM(5×1 min), DMF (5×1 min), MeOH (5×1 min), DMF (5×1 min), THF (5×1 min),DMF (5×1 min), DCM (4×1 min), ether (3×1 min), and was dried undervacuum.

1.17 g of the dry peptidyl-resin were treated with 15 mL ofTFA-^(i)Pr₃Si—H₂O (90:5:5) for 2 hours at room temperature. Thefiltrates were collected on cold diethyl ether (100 mL), centrifuged for5 minutes at 4000 rpm and the ether solution was decanted. The washingswith ether were repeated 5 times. The final precipitate was dried undervacuum.

The analysis by HPLC in a gradient from 5 to 95% of MeCN (+0.07% TFA) inH₂O (+0.1% TFA) showed a retention time of 19.3 minutes and a puritygreater than 70%. Its molecular weight was determined by ES-MS[(M+H)_(+theoretical) 688.45, (M+H)⁺ _(exp) 688.7].

Example 3: (Prophetic) Obtaining Ac-βAla-His-Ser-His-NH—(CH₂)₉—CH₃

2.0 g of Fmoc-L-His(Trt)-OH (3.23 mmol, 1 equiv) dissolved in 20 mL ofDCM to which 500 μL of DIEA (1.1 mmol, 0.33 equiv) have been added wereincorporated to dry 2-chlorotrityl resin (2.0 g, 3.3 mmol). It was leftstirring for 5 minutes, after which 1 mL of DIEA (2.2 mmol, 0.67 equiv)were added. It was allowed to react for 40 minutes. The remainingchloride groups were blocked by treatment with 1.6 mL of MeOH.

The amino terminal Fmoc group was deprotected on 1 mmol of theaminoacyl-resin as described in general methods and 1.95 g ofFmoc-L-Ser(tBu)-OH (5 mmol, 5 equiv) were incorporated in the presenceof DIPCDI (770 μL, 5 mmol, 5 equiv) and HOBt (770 mg, 5 mmol, 5 equiv)using DMF as a solvent for 1 hour. The resin was subsequently washed asdescribed in the general methods and the treatment for deprotecting theFmoc group was repeated to incorporate the next amino acid. By followingthe described protocols, 3.09 g of Fmoc-L-His(Trt)-OH (5 mmol, 5 equiv)and 1.60 g of Fmoc-βAla-OH (5 mmol, 5 equiv) were coupled sequentiallywith the presence in each coupling of 770 mg of HOBt (5 mmol, 5 equiv)and 770 μL of DIPCDI (5 mmol, 5 equiv).

The N-terminal Fmoc group was deprotected as described in generalmethods, and the peptidyl-resin was treated for 30 minutes with 2.36 mLof acetic anhydride (25 mmol, 25 equiv) in the presence of 4.28 mL ofDIEA (25 mmol, 25 equiv) using DMF as a solvent, it was washed with DMF(5×1 minutes), DCM (4×1 minutes), diethyl ether (4×1 minutes) and driedunder vacuum.

The completely protected peptide[Ac-βAla-L-His(Trt)-L-Ser(tBu)-L-His(Trt)-OH] was obtained by treatmentfor 5 minutes of the peptidyl-resin, previously dried under vacuum inthe presence of KOH, with a 3% solution of TFA in DCM. The filtrateswere collected on cold diethyl ether and the treatment was repeatedthree times. The ether solutions were rotary evaporated to dryness atroom temperature, the precipitate was resuspended in 50% MeCN in H₂O andlyophilized. The raw product obtained was analyzed by HPLC in a gradientfrom 5 to 95% of MeCN (+0.07% TFA) in H₂O (+0.1% TFA), showing aretention time of 24.1 minutes and a purity greater than 88%. Itsmolecular weight was determined by ES-MS [(M+H)⁺ _(theoretical) 1034.2,(M+H)⁺ _(exp) 1034.0].

380 mg of Ac-βAla-L-His(Trt)-L-Ser(tBu)-L-His(Trt)-OH (367 μmol) wereweighed in a balloon, 350 mg of decylamine (3 equiv) and 30 mL ofanhydrous DMF were added. 120 μL of DIPCDI (2 equiv) were added, and itwas allowed to react with magnetic stirring at 47° C. The reaction wascontrolled by means of HPLC by the disappearance ofAc-βAla-L-His(Trt)-L-Ser(tBu)-L-His(Trt)-OH, being completed in 2.5hours. The solvent was evaporated to dryness and was coevaporated twicewith DCM. The obtained residue[Ac-βAla-L-His(Trt)-L-Ser(tBu)-L-His(Trt)-NH—(CH₂)₉—CH₃] was resuspendedin 50 mL of a mixture of TFA-DCM-anisole (49:49:2) and was allowed toreact for 30 minutes at room temperature. 250 mL of cold diethyl etherwas added, the solvent was rotary evaporated and two additionalcoevaporations were carried out with ether. The residue was dissolved ina 50% mixture of MeCN in H₂O and lyophilized.

The analysis by HPLC in a gradient from 5 to 85% of MeCN (+0.07% TFA) inH₂O (+0.1% TFA) indicated a retention time of 16.6 minutes and a puritygreater than 71%. Its molecular weight was determined by ES-MS [(M+H)⁺_(theoretical) 632.4, (M+H)⁺ _(exp) 632.6].

Example 4 Obtaining Ac-βAla-His-Ser-His-OH

The peptide of Example 4 was obtained by following the same syntheticprotocol as in Example 1 (amounts, solvents, excesses and reactants),but incorporating Fmoc-L-Ser(tBu)-OH (8.43 g, 22 mmol) instead ofFmoc-L-Cys(Trt)-OH as the second amino acid. Once the synthesis wascompleted, the resin was washed with DMF (5×1 minutes), DCM (4×1minutes), diethyl ether (4×1 minutes) and dried under vacuum.

The peptide was released from the solid support by following the samesynthetic protocol as in Example 1 (amounts, solvents, excesses andreactants).

The analysis by HPLC in a gradient from 0 to 10% of MeCN (+0.07% TFA) inH₂O (+0.1% TFA) indicated a purity greater than 90% and its molecularweight was determined by ES-MS [(M+H)⁺ _(theoretical) 493.22, (M+H)⁺_(exp) 493.2].

Example 5 Preparation of a Cosmetic Composition ContainingAc-βAla-His-Ser-His-OH

The following formulation was prepared as described in the presentinvention:

The oils, surfactants and carbomers were weighed in a sufficiently largereactor. The peptide was weighed in another reactor, it was dissolved inwater and the preservative (Phenonip®) was added to it. The peptidesolution was poured on to the oil solution with constant vigorousstirring. Once the addition is over, the pH is adjusted to 6.5-7.0 withtriethanolamine.

% BY INGREDIENT (INCI Nomenclature) WEIGHT ACRYLATES/C10-30 ALKYLACRYLATES 0.3 CROSSPOLYMER CARBOMER 0.3 C12-15 ALKYL BENZOATE 17.6ETHYLHEXYL COCOATE 2.4 POLYACRYLAMIDE, C13-14 1.0 ISOPARAFFIN, LAURETH-7AQUA (WATER) q.s.p. 100 PHENONIP ® 0.5 Ac-βAla-His-Ser-His-OH 0.01TRIETHANOLAMINE 0.7

Example 6 Preparation of a Cosmetic Composition ContainingAc-βAla-His-Cys-His-OH

The following formulation was prepared as described in the presentinvention:

The oils, waxes, silicones and carbomers were weighed in a sufficientlylarge reactor. The mixture is heated to 65-70° C. to melt the waxes. Theglycerin was weighed in another reactor, it was resuspended in water andthe preservative (Phenonip®) and the triethanolamine were added. Themixture was heated to 65-70° C. with constant vigorous stirring or bymeans of applying shear force. Once the addition was over, it wasallowed to cool with slow stirring and when the mixture is at 40° C., anaqueous Ac-βAla-His-Cys-His-OH solution was added. The cream is allowedto cool to room temperature and the pH is corrected with triethanolamineif necessary.

INGREDIENT (INCI Nomenclature) % BY WEIGHT MINERAL OIL 10.0 STEARIC ACID3.0 BEESWAX 2.0 DIMETHICONE 0.2 CARBOMER 0.2 GLYCERIN 3.0 AQUA (WATER)q.s.p. 100 PHENONIP ® 0.5 Ac-βAla-His-Cys-His-OH 0.005 TRIETHANOLAMINE2.0

Example 7

A clinical study of the cosmetic composition described in Example 5conducted in 20 subjects with bags formed under the eyes showed that thecomposition can reduce the size of the bags formed under the eyes. Thesubjects were instructed to apply the cosmetic composition in the eyecontour area with a soft massage once a day for two months. Objectivemeasurements of the degree of skin hydration by means of a corneometer(Skinlab®) and of skin elasticity by means of an elastometer werecarried out at time 0 and 15, 30 and 60 days after the start of thetreatment, and the size of the under-eye bags was also observed by adermatologist.

The quantification of the results showed a 5.8% increase of thehydration index as well as a 35% increase of the elasticity index after60 days of treatment. A subjective evaluation by the dermatologist ofthe appearance of the eye bags according to Friedman's test [Cristoni A.(2001) La significativitá del risultato sperimentale. Quali test usare?Cosmetic News 130, January/February pp 30-32] confirmed that 70% of thevolunteers experiences a reduction of under-eye bags in only 15 days,which percentage rose to 95% of the volunteers after 60 days oftreatment (30% slight reduction, 30% discreet reduction and 35%significant reduction).

According to a first aspect, the present invention relates to a peptideof general formula (I):

its stereoisomers, its cosmetically and dermopharmaceutically acceptablesalts and mixtures thereof, wherein:

-   -   X is selected from the group formed by cystenyl cysteinyl,        seryl, threonyl and aminobutyryl;    -   R₁ is selected from the group formed by H or alkyl, aryl,        aralkyl, and acyl group and,    -   R₂ is selected from the group formed by amino, hydroxy and        thiol, substituted or non-substituted with aliphatic or cyclic        groups.

According to a second important aspect, in the peptide of generalformula (I) R₁ is preferably saturated or unsaturated, branched orcyclic, linear C₂ to C₂₄ acyl.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) R₂ is preferably amino or hydroxy, substituted ornon-substituted with saturated or unsaturated, branched or cyclic,linear aliphatic C₁ to C₂₄ groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is H and R₂ is amino,substituted or non-substituted with methyl or ethyl or dodecyl orhexadecyl groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is acetyl and R₂ isamino, substituted or non-substituted with methyl or ethyl or dodecyl orhexadecyl groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is palmitoyl and R₂ isamino, substituted or non-substituted with methyl or ethyl or dodecyl orhexadecyl groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is H and R₂ is hydroxy,substituted or non-substituted with methyl or ethyl or dodecyl orhexadecyl groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is acetyl and R₂ ishydroxy, substituted or non-substituted with methyl or ethyl or dodecylor hexadecyl groups.

According to an important aspect of the invention, in the peptide ofgeneral formula (I) X is preferably L-seryl, R₁ is palmitoyl and R₂ ishydroxy, substituted or non-substituted with methyl or ethyl or dodecylor hexadecyl groups.

According to another important aspect, the present invention relates toa process for obtaining a peptide of general formula (I) based onsolid-phase peptide synthesis.

According to another important aspect, the present invention relates toa process for obtaining a peptide of general formula (I) usingprotective groups selected from the group formed by Fmoc/tButyl,Fmoc/trityl and Fmoc/allyl.

According to another important aspect, the present invention relates toa cosmetic or dermopharmaceutical composition comprising a cosmeticallyor dermopharmaceutically effective amount of at least one peptide offormula (I) and at least one cosmetically or demopharmaceuticallydermopharmaceutically acceptable excipient or adjuvant.

According to another important aspect, the present invention relates toa cosmetic or dermopharmaceutical composition containing a peptide ofgeneral formula (I) incorporated to a cosmetically ordemopharmaceutically dermopharmaceutically acceptable carrier selectedfrom the group formed by liposomes, millicapsules, microcapsules andnanocapsules, sponges, millispheres, microspheres, nanospheres,milliparticles, microparticles and nanoparticles.

According to another important aspect, the present invention relates toa cosmetic or dermopharmaceutical composition having a formulationselected from the group formed by oil-in-water emulsions, water-in oilemulsions, creams, milks, lotions, gels, ointments, liniments, serums,mousses, balms, foams, body oils, soaps, bars, pencils and sprays.

According to another important aspect, the present invention relates toa cosmetic or dermopharmaceutical composition containing a peptide ofgeneral formula (I) incorporated to solid supports selected from thegroup formed by towelletes, hydrogels, patches and face masks.

According to another important aspect, the present invention relates toa cosmetic or dermopharmaceutical composition containing a peptide ofgeneral formula (I) incorporated to make-up line products selected fromthe group formed by concealers, make-up foundations, lotions, andmake-up removal lotions.

According to another important aspect, the present invention relates tothe use of a peptide of formula (I) in the manufacture of a cosmetic ordermopharmaceutical composition for the treatment of skin.

According to another important aspect, the present invention relates tothe use of a peptide of formula (I) in the manufacture of a cosmetic ordermopharmaceutical composition for the treatment of facial skin.

According to another important aspect, the present invention relates tothe use of a peptide of formula (I) in the manufacture of a cosmetic ordermopharmaceutical composition for reducing or removing bags formedunder the lower eye contour.

The invention claimed is:
 1. A peptide of general formula (I):

its stereoisomers, mixtures thereof, and its cosmetically anddermopharmaceutically acceptable salts, wherein: X is selected from thegroup formed by cystenyl consisting of cysteinyl, seryl, threonyl andaminobutyryl; R₁ is selected from the group formed by consisting of H orand alkyl, aryl, aralkyl and acyl group groups; and R₂ is selected fromthe group formed by consisting of amino, hydroxy and thiol, substitutedor non-substituted with aliphatic or cyclic groups.
 2. A peptideaccording to claim 1, wherein R₁ is saturated or unsaturated, branchedor cyclic, linear C₂ to C₂₄acyl C₂₄ acyl.
 3. A peptide according toclaim 1, wherein R₂ is amino or hydroxy, substituted or non-substitutedwith saturated or unsaturated, branched or cyclic, linear C₁ to C₂₄aliphatic groups.
 4. A peptide according to claim 1, wherein X is L-Ser,R₁ is H and R₂ is amino, substituted or non-substituted with methyl orethyl or dodecyl or hexadecyl groups.
 5. A peptide according to claim 1,wherein X is L-Ser, R₁ is acetyl and R₂ is amino, substituted ornon-substituted with methyl or ethyl or dodecyl or hexadecyl groups. 6.A peptide according to claim 1, wherein X is L-Ser, R₁ is palmitoyl andR₂ is amino, substituted or non-substituted with methyl or ethyl ordodecyl or hexadecyl groups.
 7. A peptide according to claim 1, whereinX is L-Ser, R₁ is H and R₂ is hydroxy, substituted or non-substitutedwith methyl or ethyl or dodecyl or hexadecyl groups.
 8. A peptideaccording to claim 1, wherein X is L-Ser, R₁ is acetyl and R₂ ishydroxy, substituted or non-substituted with methyl or ethyl or dodecylor hexadecyl groups.
 9. A peptide according to claim 1, wherein X isL-Ser, R₁ is palmitoyl and R₂ is hydroxy, substituted or non-substitutedwith methyl or ethyl or dodecyl or hexadecyl groups.
 10. A cosmetic ordermopharmaceutical composition comprising a cosmetically ordemopharmaceutically dermopharmaceutically acceptable excipient oradjuvant; and an effective amount of the peptide of claim
 1. 11. Acosmetic or dermopharmaceutical composition according to claim 10,wherein the composition contains at least one peptide of general formula(I) incorporated to a cosmetically or demopharmaceuticallydermopharmaceutically acceptable carrier selected from the group formedby consisting of liposomes, millicapsules, microcapsules, nanocapsules,sponges, millispheres, microspheres, nanospheres, milliparticles,microparticles and nanoparticles.
 12. A cosmetic or dermopharmaceuticalcomposition according to claim 10, wherein it has a formulation selectedfrom the group formed by consisting of oil-in-water emulsions,water-in-oil emulsions, milks, lotions, gels, ointments, balms, foams,body oils, soaps, bars, pencils, sprays, creams, liniments, unguents,serums and mousses.
 13. A cosmetic or dermopharmaceutical compositionaccording to claim 10, wherein it contains at least one peptide ofgeneral formula (I) incorporated to solid supports selected from thegroup formed by consisting of towelletes, hydrogels, patches and facemasks.
 14. A cosmetic or dermopharmaceutical composition according toclaim 10, wherein it contains at least one peptide of general formula(I) incorporated to make-up line products selected from the group formedby consisting of concealers, make-up foundations, lotions, and make-upremoval lotions.
 15. A method of treating facial skin in need skin,comprising administering to the skin an effective amount of a peptide ofclaim 1 for reducing bags formed under the lower eye contour.
 16. Themethod of claim 15, wherein a cosmetic or dermopharmaceuticalcomposition comprises the peptide of general formula (I).
 17. A peptideof general formula (I):

its stereoisomers, mixtures thereof, and its cosmetically anddermopharmaceutically acceptable salts, wherein: X is selected from thegroup formed by cystenyl consisting of cysteinyl, seryl, threonyl andaminobutyryl; R₁ is selected from the group formed by consisting of H orand saturated or unsaturated, branched or cyclic, linear C₂ to C₂₄acyl;and R₂ is selected from the group formed by consisting of amino, hydroxyand thiol, substituted or non-substituted with aliphatic or cyclicgroups.
 18. A cosmetic or dermopharmaceutical composition, comprising acosmetically or demopharmaceutically acceptable excipient or adjuvant;and an effective amount of the peptide of claim
 17. 19. A method oftreating facial skin in need skin, comprising administering to the skinan effective amount of the peptide of claim 17 for reducing bags formedunder the lower eye contour.
 20. A peptide according to claim 1, whereinR₁ is acetyl.
 21. A peptide according to claim 1, wherein R₁ ispalmitoyl.
 22. A peptide according to claim 1, wherein R₁ is —H.
 23. Apeptide according to claim 1, wherein R₂ is —OH.
 24. A peptide accordingto claim 1, wherein R₂ is —NH₂.
 25. A peptide according to claim 1,wherein R₂ is —NH—(CH₂)₉—CH₃.
 26. A peptide according to claim 1, X isseryl.
 27. A peptide according to claim 1, wherein X is aminobutyryl.28. A peptide according to claim 1, wherein X is cysteinyl.
 29. Apeptide according to claim 7, wherein X is L-Ser, R₁ is H, and R₂ ishydroxy.
 30. A peptide according to claim 8, wherein X is L-Ser, R₁ isacetyl, and R₂ is hydroxy.
 31. A peptide according to claim 1, wherein Xis L-Ser, R₁ is palmitoyl and R₂ is hydroxy.
 32. A cosmetic ordermopharmaceutical composition according to claim 10, wherein saidexcipient or adjuvant is selected from the group consisting of emulsionagents, emollients, organic solvents, skin conditioners, gellingpolymers, thickeners, softeners, anti-wrinkle agents, whitening agents,compounds capturing free radicals, anti-oxidizing compounds, compoundsstimulating collagen synthesis, compounds stimulating elastin synthesis,compounds inhibiting collagen degradation, compounds stimulatingfibroblast proliferation, compounds stimulating keratinocyteproliferation, compounds stimulating keratinocyte differentiation, skinrelaxing compounds, compounds stimulating glycosaminoglycan synthesis,firming compounds, compounds acting on capillary circulation, compoundsacting on cell metabolism, compounds stimulating melanin synthesis,preservatives, perfumes, chelating agents, plant extracts, essentialoils, marine extracts, cell extracts, and sunscreens.